A new species of slender flatworm in the genus Eucestoplana and a record of E. cf. cuneata (Platyhelminthes, Polycladida) from the Okinawa Islands, Japan, with an inference of their phylogenetic positions within Cestoplanidae Author Tsuyuki, Aoi https://orcid.org/0000-0002-6001-0679 Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060 - 0810, Japan tykamsp0430@gmail.com Author Oya, Yuki https://orcid.org/0000-0001-6862-6868 Creative Research Institute, Hokkaido University, Sapporo 001 - 0021, Japan Author Kajihara, Hiroshi https://orcid.org/0000-0001-6510-9355 Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060 - 0810, Japan text Zoosystematics and Evolution 2023 2023-07-05 99 2 363 373 http://dx.doi.org/10.3897/zse.99.102604 journal article http://dx.doi.org/10.3897/zse.99.102604 1860-0743-2-363 D7ACA6364B0346F4AF77D5DEC8EB7084 834F4D6012375035847C365EADB50A21 Eucestoplana ittanmomen sp. nov. Figs 4 , 5 Material examined. Holotype : Japan •1; Okinawa Prefecture , the Okinawa Islands , Kouri Island , Tokei Beach ; 26°42.86'N , 128°1.108'E ; intertidal gravelly sediments; 11 Aug. 2021 ; A. Tsuyuki and Y. Oya leg.; sagittal sections (6 slides); GenBank : LC740490 (16S), LC740492 (18S), and LC740495 (28S); ICHUM 8443 . Paratype : Japan •1; same data as for holotype; sagittal sections (4 slides); ICHUM 8444 . Type locality. Japan, Okinawa Prefecture, Kunigami, Nakijin, Kouri Island, Tokei Beach ( 26°42.86'N , 128°1.108'E ). Diagnosis. Body slender and elongated; anterior margin rounded; dorsal surface translucent white without any color pattern; pair of eyespot-clusters distributed along midline in front of brain; penis papilla with heavily sclerotized stylet; penis sheath dome-shaped with external epithelium covered with cilia; cilia absent in inner wall of male atrium; adhesive organ present at posterior end of body. Description of holotype. Body slender and elongated, 26 mm long and 0.75 mm wide in living state (Fig. 4A ); anteriorly rounded, spreading like fan; posteriorly tapered. Dorsal surface smooth, translucent, without any color pattern. Ventral surface translucent. Tentacles absent. Pair of eyespot-clusters, each composed of 12-14 eyespots (12 on left; 14 on right), distributed along midline in front of brain (Fig. 4B ), spreading out in fan shape anteriorly. Intestine highly branched without anastomosing, spreading throughout body, not reaching body margin. Pharynx ruffled, 1.94 mm long, situated on last fourth of body (Fig. 4A, C ). Mouth opening at last third of pharyngeal pouch (Fig. 4C ). Male gonopore opening at last ninth of body (Fig. 4A ). Female gonopore situated posterior to male gonopore. Male copulatory apparatus consisting of true seminal vesicle, interpolated prostatic vesicle, and penis papilla with stylet (Fig. 4A ). Testicular follicles arranged in single, lateral, longitudinal row on each side, about half length of body, running anteriorly from area in front of pharynx (Fig. 4A ). Pair of sperm ducts separately entering proximal end of seminal vesicle; each duct forming spermiducal vesicle before entering seminal vesicle (Fig. 5A, B ). Seminal vesicle extending posteriorly, about 700 µm long and 90 µm wide at its widest point, posteriorly turning 180° right in front of female copulatory apparatus before running anteriorly to lead to ejaculatory duct at position of proximal end of penis stylet; thick muscular wall, about 19 µm thickness, coating seminal vesicle and ejaculatory duct (Fig. 5A, B ). Prostatic vesicle oval, elongated, with about 18-µm thick muscular wall, lined with thick glandular epithelium; distal end of prostatic vesicle forming penis papilla (Fig. 5A, C ). Penis papilla with wedged, strongly sclerotized stylet (131 µm long), projecting into male gonopore (Fig. 5A, D ). Penis sheath dome-shaped, about 184 µm wide at its widest point, housing penis stylet (Fig. 5A, C, D ); external epithelium being exposed to male atrium, former being lined with cilia (Fig. 5C, D ); penis pocket lined with non-ciliated epithelium. Male atrium lined with thin epithelium without cilia (Fig. 5C, D ). Male gonopore about 27 µm deep. Female copulatory organ lacking Lang's vesicle. Pair of oviducts running posteriorly, then connecting to proximal end of vagina independently. Vagina narrow, curved dorsoventrally, lined with ciliated epithelium, leading to female gonopore via narrow cement pouch (Fig. 5A, E ). Numerous cement glands releasing their contents into cement pouch (Fig. 5E ). Adhesive organ located at posterior end of body. Figure 4. Eucestoplana ittanmomen sp. nov., holotype (ICHUM 8443). A. Whole animal in living state, dorsal view; B. Magnification of anterior body, dorsal view, showing eyespot distribution; C. Photomicrograph of sagittal section (anterior to the right), showing pharynx and mouth. Abbreviations: mo - mouth; ph - pharynx; te - testicular follicle; ♀ - female copulatory apparatus; ♂ - male copulatory apparatus. Scale bars: 1 mm ( A , B ); 100 µm ( C ). Description of paratype. Due to lack of anterior part of body, body length, width and eyespot arrangements unknown. Body coloration same as holotype. Pharynx ruffled, 1.27 mm in length; mouth opening at posterior region of pharyngeal pouch. Male copulatory apparatus composed of elongate seminal vesicle, interpolated prostatic vesicle, and penis papilla with wedged stylet (106 µm long); penis stylet slenderer than that of holotype. Penis sheath dome-shaped, with external epithelium ciliated; numerous eosinophilic glands piercing distal part of penis sheath. Male atrium covered with non-ciliated epithelium. Female copulatory apparatus same as holotype except for shape of cement pouch being more expanded than that of holotype. Adhesive organs present at posterior end of body (Fig. 5F ). Figure 5. Eucestoplana ittanmomen sp. nov., schematic diagram ( A ) and photomicrographs of sagittal sections ( B - F ) (anterior to the right). A. ICHUM 8443 (holotype), male and female copulatory apparatuses; B-D. ICHUM 8443 (holotype), male copulatory apparatus; E. ICHUM 8443 (holotype), female copulatory apparatus; F. ICHUM 8444 (paratype), adhesive organ. Abbreviations: ad - adhesive organ; cg - cement glands; cp - cement pouch; ed - ejaculatory duct; fg - female gonopore; ma - male atrium; mg - male gonopore; po - penis pouch; pp - penis papilla; ps - penis sheath; pv - prostatic vesicle; spv - spermiducal vesicle; st -stylet; sv - seminal vesicle; va - vagina. Scale bars: 100 µm ( A - F ). Etymology. The specific name ittanmomen (Ittan-momen) is a Japanese noun, representing the name of one of the "yokai" (a class of supernatural entities and spirits in Japanese folklore). It is named after the long and narrow cloth-like white body of the flatworm, which evokes the similar-looking yokai, Ittan-momen. Distribution. To date, only from the Okinawa Islands, Japan. Remarks. Our specimens belong to Eucestoplana based on the following characteristics: i ) the evident sclerotized penis stylet and ii ) a female copulatory apparatus without any accessory ducts or Lang's vesicle. Eucestoplana ittanmomen sp. nov. can be easily distinguished from E. meridionalis by the following characteristics: i ) translucent body, ii ) fewer eyespots distributed only anterior to the brain, and iii ) the presence of the adhesive organ (Table 3 ). Our new species is most similar to E. cuneata in having the following characteristics: i ) around 30 eyespots distributed only anterior to the brain, ii ) a wedge-shaped stylet, and iii ) the adhesive organ located on the posterior end of the body. However, E. ittanmomen sp. nov. is differentiated from E. cuneata by the following characteristics: i ) the shape of the penis sheath (dome-shaped in E. ittanmomen sp. nov.; cone-shaped in E. cuneata ), ii ) the arrangement of the cilia in the inner wall of the male atrium (only present along the outside of the penis sheath in E. ittanmomen sp. nov.; surrounding the whole male atrium in E. cuneata ), and iii ) the stylet length (106-131 µm in E. ittanmomen sp. nov.; 70 µm in E. cuneata ). Eucestoplana ittanmomen sp. nov. can be also distinguished from E. cf. cuneata collected from the same locality by the same morphological differences as mentioned above. In addition, the genetic distance for 16S and 28S sequences between them could support that the two entities are likely to be genetically independent. The values for 16S (3.153%-3.378%) were much larger than the three interspecific values 0.5-1.8%, which were observed among three species of Notocomplana ( N. hagiyai , N. japonica , and N. koreana ) ( Oya and Kajihara 2017 ). The p -distance between the 28S sequences of E. ittanmomen sp. nov. and E. cf. cuneata was also much larger than that between C. salar and C. techa , which are clearly different species because of their morphological difference (Table 2 ). Table 3. Comparison of the selected characteristics among the known Eucestoplana species and our new species.

-	E. cuneata	E. ittanmomen sp. nov.	E. meridionalis
Body length (mm)	10a	26	20
Body width (mm)	?(slender, ribbon-shaped)	0.7	3
Anterior body shape	Rounded	Rounded	Slightly pointed
Eyespots	35-40a, only anterior to the brain	About 20-30, only anterior to the brain	Numerous, distributed around brain
Dorsal coloration	? a; translucent whiteb	Translucent white	Chocolate-brown
Dorsal color pattern	? a	Absent	Absent
Mouth position	Near posterior end of pharynx	Near posterior end of pharynx	In posterior region of pharyngeal cavity
Seminal vesicle	Elongate, bending 180°	Elongated, bending 180° at position posterior to female reproductive organ	Elongate-oval
Stylet	70-µm long; wedge-shaped	106-131-µm long; wedge-shaped	Present
Penis sheath	Cone-shaped	Dome-shaped	Cone-shaped
Cilia along inner wall of male atrium	Surrounding the whole male atrium	Only present along the outside of the penis sheath	?
Adhesive organ	Present	Present	Absent
Distribution	The Galapagos Islandsa; Fijib	The Okinawa Islands, Japan	South Australia
Reference	a Sopott-Ehlers and Schmidt (1975) ; b Tajika et al. (1991)	This study	Prudhoe (1982a) ; Prudhoe (1982b)